CN111686399B

CN111686399B - Reagent for inhibiting expression of antibiotic resistance genes of bacteria under metal induction and preparation method and application thereof

Info

Publication number: CN111686399B
Application number: CN202010480068.XA
Authority: CN
Inventors: 吴平霄; 李义豪; 王慧敏; 牛文超; 王锐; 朱能武; 党志
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-05-29
Filing date: 2020-05-29
Publication date: 2022-05-24
Anticipated expiration: 2040-05-29
Also published as: CN111686399A

Abstract

The invention discloses a reagent for inhibiting the expression of antibiotic resistance genes of bacteria under metal induction, and a preparation method and application thereof. The method comprises: adding montmorillonite into water, centrifuging to get a precipitate, adding the precipitate back to the water, repeating centrifugation until the supernatant is clear, drying, sieving, adding to a solvent, and mixing to obtain the inhibitory bacteria induced by the metal Reagents for gene expression under antibiotic resistance. This reagent resists low-dose antibiotic stress by utilizing Mt to modulate the expression pattern and metabolism of bacterial antibiotic resistance genes induced by Cd ²⁺ , thereby inhibiting the expression of antibiotic resistance genes in E. coli . The results of minimum inhibitory concentration and real-time quantitative PCR also indicated that Mt could attenuate the expression of bacterial antibiotic resistance induced by Cd ²⁺ . Therefore, Mt can be used as an environmental material to alleviate the expression of bacterial antibiotic resistance genes under heavy metal stress, which is of great significance to control the contamination of ARGs caused thereby.

Description

Reagent for inhibiting expression of antibiotic resistance gene of bacteria under metal induction, preparation method and application thereof

Technical Field

The invention belongs to the fields of mineralogy and environmental microbiology, and particularly relates to a reagent for inhibiting the expression of antibiotic resistance genes of bacteria under metal induction, and a preparation method and application thereof.

Background

Antibiotic resistance is one of the most serious threats to public health. Overuse of antibiotics induces the production of bacterial Antibiotic Resistance Genes (ARGs), which become a key cause of the spread of antibiotic resistance. The ARGs are novel environmental pollutants and have great harm to environmental media, particularly soil environment.

A large number of researches show that the ARGs are induced by not only antibiotics, but also non-antibiotic substances such as heavy metals and organic matters. There is a close relationship between heavy metals and antibiotic resistance, which can enhance antibiotic resistance through a co-selection mechanism. In a heavy metal pollution area, heavy metal can induce bacteria to generate heavy metal resistance and can also induce bacteria to generate multiple antibiotic resistance, and the existence level of the ARGs is in positive correlation with the heavy metal pollution level. More noteworthy is that the content of heavy metals in the environmental medium is much higher than that of antibiotics and that heavy metals are not degraded compared to antibiotics, which will put longer-term stress on the environment. Therefore, the research on reducing the diffusion of the ARGs under the induction of the heavy metals has important significance on reducing the environmental health risks.

Clay minerals are the most common substances in the earth's surface environment, and have complex interfacial reactions with microorganisms and heavy metals in soil. On the one hand, microbial activity can promote dissolution, precipitation and transformation of minerals, thereby accelerating biogeochemical cycles and affecting the evolution of landforms and the earth ecosystem. On the other hand, the clay mineral can regulate the growth and metabolism of microorganisms, and has great influence on the distribution, activity, diversity, gene expression and transformation of the microorganisms under external pressure. However, few researchers have deeply explored from a molecular biology perspective how clay minerals regulate the antibiotic resistance gene generation process of microorganisms under heavy metal stress. The Mt has excellent biocompatibility, and can enable bacteria to have the potential of regulating gene expression mode and growth metabolic activity under heavy metal stress, so that heavy metal resistance is enhanced to relieve the environmental stress of heavy metal pollution and the environmental stress and ecological risk brought by the generation of antibiotic resistance genes under the induction of heavy metals. Therefore, the research on the antibiotic resistance expression mechanism of Mt to bacteria under the metal stress can further provide a new idea and a new technology for promoting heavy metal pollution treatment and relieving the expression of ARGs.

Disclosure of Invention

In order to solve the problem of pollution of antibiotic resistance genes, the invention provides a reagent for inhibiting the expression of antibiotic resistance genes of bacteria under the induction of metals, and a preparation method and application thereof.

The purpose of the invention is realized by at least one of the following technical solutions.

The invention provides a reagent for inhibiting the expression of antibiotic resistance genes of bacteria under the induction of metal, which comprises montmorillonite.

Further, the montmorillonite (Mt) needs to be sterilized at high temperature before use, and the temperature is 100-120 ℃.

Further, the concentration of Mt is 10-20gL^-1。

Further, the pH of the reagent is 6.0-8.0.

Further, the metal is induced to Cd²⁺Ion induced environment.

Further, the Cd²⁺The concentration of the ions is 16-128 mug.mL^-1。

The invention provides a method for preparing an agent for inhibiting the expression of an antibiotic resistance gene of bacteria under the induction of metal, which comprises the following steps:

(1) adding montmorillonite into water, centrifuging, removing supernatant, collecting precipitate, adding the precipitate into water again, repeating the centrifuging step until the supernatant is clear, oven drying, sieving, and sterilizing at high temperature to obtain pretreated montmorillonite;

(2) And (2) adding the pretreated montmorillonite obtained in the step (1) into a solvent, and uniformly mixing to obtain the reagent for inhibiting the expression of the antibiotic resistance genes of the bacteria under the induction of metals.

Further, the rotation speed of the centrifugal treatment in the step (1) is 8000-10000 rpm; the drying temperature in the step (1) is 55-65 ℃.

Further, the size of the sieve holes of the sieve in the step (1) is 200 meshes.

Further, the temperature of the high-temperature sterilization treatment in the step (1) is 100-120 ℃, and the time of the high-temperature sterilization treatment is 30-60 min.

Further, the solvent in the step (2) is deionized water.

The invention provides an application of a reagent for inhibiting the expression of a bacterial antibiotic resistance gene in the preparation of an antibacterial agent.

The invention utilizes the influence of Mt on the growth, the propagation and the metabolism of microorganisms, regulates the gene expression mode of bacteria, resists metal-induced antibiotic stress under low dose, further reduces the possibility of resisting the expression of the ARGs, and has important significance for controlling the ARGs pollution caused by the ARGs.

The effect of the agent for inhibiting the expression of the bacterial antibiotic resistance gene provided by the invention can be verified through the following experiments. The validation experiment included the following steps.

1) Mt pretreatment:

Washing Mt raw soil with deionized water, centrifuging at 8000rpm to remove soluble impurities on the surface, and treating for multiple times until the deionized water is clear to obtain a precipitate which is pure Mt solid. Then the sample is put into an oven at 60 ℃ for blast drying, sieved by a 200-mesh sieve and subjected to high-temperature sterilization treatment (100 ℃ and 120 ℃) for subsequent analysis.

2) And (3) culturing bacteria:

the bacteria were wild type E.coli Escherichia coli ATCC25922(E.coli) cultured in MHB medium at 37 ℃ under pH 7.0 and shaking at 200 rpm.

3) Gradient concentration induction of heavy metals on bacteria in the presence or absence of Mt

The heavy metal is cadmium (Cd)²⁺) Starting from the sub-inhibitory concentration of bacteria, Cd is utilized²⁺Coli induces antibiotic resistance in antibiotic-susceptible bacteria e. Coli to 30mL of Cd at a concentration of 16. mu.g.mL in the presence and absence of Mt at a ratio of 1:100, respectively^-1、32μg·mL^-1、64μg·mL^-1And 128. mu.g.mL^-1Was cultured for three generations in fresh NB medium at 37 ℃ and pH 7.0 at 200rpm for 24 hours each. Then transferred to the medium under the same conditions for 3 passages, each for 24 h.

4) Analysis of the Minimum Inhibitory Concentration (MIC) of bacteria for heavy metals and antibiotics

The MIC of heavy metals was determined by the microbulking method. Diluting the tested bacteria with LB culture medium and dispersing to Cd²⁺The concentration was 16. mu.g/mL^-1、32μg·mL^-1、48μg·mL^-1、64μg·mL^-1、80μg·mL^-1、98μg·mL^-1、112μg·mL^-1、128μg·mL^-1And ensuring that the number of bacteria per well is 10⁴And (4) CFU. Subsequently, the 96-well plate was placed in a constant temperature incubator (37 ℃) and incubated for 24 hours, and then the results were checked. The determination of the MIC value is the concentration at which no bacterial growth begins to occur in the first line.

In addition, the MIC of antibiotics was also determined by broth microdilution. The bacteria to be tested (after Cd or Cd-Mt induction) were diluted with MHB medium and dispersed to contain 0.5. mu.g.mL^-1、1μg·mL^-1、2μg·mL^-1、4μg·mL^-1、8μg·mL^-1、16μg·mL^-1、32μg·mL^-1、64μg·mL^-1、128μg·mL^-196-well plates with various antibiotic concentrations and with a bacterial count of 10 per well⁴And (4) CFU. Subsequently, the 96-well plate was placed in a constant temperature incubator (37 ℃) and incubated for 24 hours, and then the results were checked. The determination of the MIC value is the concentration at which no bacterial growth begins to occur in the first line.

5) Cd induced changes in the MIC of bacteria compared to the presence and absence of Mt.

And (3) acquiring the change of the Cd-induced bacteria ARGs under the conditions that the Mt exists and the Mt does not exist by utilizing real-time fluorescent quantitative PCR. Expression levels of antibiotic-associated genes such as penicillin (e.g., mrdA, mrcA, dacB/D), tetracycline (e.g., accC), erythromycin (e.g., suhB), and chloramphenicol (e.g., tktA). And the expression conditions of genes related to multidrug resistance/multiple antibiotic resistance/antibiotic response (such as mdtQ, marB/R, arnA/B/C/D) and a large number of toxin efflux related genes (such as emrA, mprA, ydhC, marA) of the bacteria under Cd stress.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the reagent provided by the invention resists low-dose antibiotic stress by using Mt to regulate the expression mode and metabolism of bacteria under the induction of heavy metals, so that the expression of gene mutation is inhibited, and the expression of antibiotic resistance genes is reduced; the preparation method provided by the invention has the advantages of simple steps, no need of special equipment, easily obtained raw materials, low investment and the like.

Drawings

FIG. 1 is a flow chart of the preparation of a reagent for inhibiting the expression of antibiotic resistance genes induced by bacterial metals.

Detailed Description

The following examples are included to further illustrate the practice of the invention, but are not intended to limit the practice or protection of the invention. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.

Example 1

A method of preparing an agent that inhibits the expression of a metal-induced antibiotic resistance gene in a bacterium, comprising the steps of:

(1) adding montmorillonite into water, centrifuging at 8000rpm, removing supernatant, collecting precipitate, adding the precipitate into water, repeating centrifuging until the supernatant is clear, oven drying at 55 deg.C, sieving with 200 mesh sieve mesh, and sterilizing at 100 deg.C for 60min to obtain pretreated montmorillonite;

(2) And (2) adding the pretreated montmorillonite obtained in the step (1) into deionized water serving as a solvent, and uniformly mixing to obtain the reagent for inhibiting the expression of the bacterial antibiotic resistance genes. In the agent for inhibiting the expression of antibiotic resistance genes in bacteria induced by metals obtained in example 1, the concentration of montmorillonite is 8g L^-1The pH of the reagent was 6.0.

Example 2

(1) adding montmorillonite into water, centrifuging at 9000rpm, removing supernatant, collecting precipitate, adding the precipitate into water again, repeating centrifuging until the supernatant is clear, oven drying at 60 deg.C, sieving with 200 mesh sieve, and sterilizing at 110 deg.C for 50min to obtain pretreated montmorillonite;

(2) and (2) adding the pretreated montmorillonite obtained in the step (1) into solvent deionized water, and uniformly mixing to obtain the reagent for inhibiting the expression of the bacterial antibiotic resistance gene. In the agent for inhibiting the expression of antibiotic resistance genes in bacteria induced by metals obtained in example 1, the concentration of montmorillonite was 12g L ^-1The pH of the reagent was 7.0.

Example 3

(1) adding montmorillonite into water, centrifuging at 10000rpm, removing supernatant, collecting precipitate, adding the precipitate into water again, repeating the centrifuging step until the supernatant is clear, oven drying at 65 deg.C, sieving with 200 mesh sieve, and sterilizing at 120 deg.C for 30min to obtain pretreated montmorillonite;

(2) and (2) adding the pretreated montmorillonite obtained in the step (1) into solvent deionized water, and uniformly mixing to obtain the reagent for inhibiting the expression of the bacterial antibiotic resistance gene. In the agent for inhibiting the expression of antibiotic resistance genes in bacteria induced by metals obtained in example 1, the concentration of montmorillonite was 16g L^-1The pH of the reagent was 8.0.

Example 4

Effect of Cd-induced e.coli on ampicillin ARGs expression in presence of Mt was verified (see scheme 1).

The strain after 12 hours of activation was adjusted to 2.5MCF (1MCF ═ 3x 10) using a bacterial turbidimeter⁸CFU·mL^-1) To ensure that the number of bacteria used in subsequent experiments is equal. The activated strains were added to Mt (8 gL) at a ratio of 1:100, respectively ^-1) And 30mL Cd with no Mt present²⁺The concentration was 16. mu.g/mL^-1Culturing for three generations in fresh LB culture medium (with sub-bacteriostasis concentration) for 24h each generation, wherein the culture conditions are 37 ℃, pH 6.0 and rotation speed of 200 rpm. Then transferred to Cd under the same conditions²⁺The concentration was 32. mu.g/mL^-1Subcultured 3 times in medium for 24h each time, and so on, and transferred to 64. mu.g.mL^-1、96μg·mL^-1、128μg·mL^-1Each passage was 3 times in the medium, each time for 24h, and the whole process was 15 days.

The test bacteria were diluted with MHB medium and dispersed to contain 0.5. mu.g.mL^-1、1μg·mL^-1、2μg·mL^-1、4μg·mL^-1、8μg·mL^-1、16μg·mL^-1、32μg·mL^-1、64μg·mL^-1、128μg·mL^-196-well ampicillin-concentration plates, and ensuring that the number of bacteria per well is 10⁴And (4) CFU. Subsequently, the 96-well plate was placed in a constant temperature incubator (37 ℃) and incubated for 24 hours, and then the results were checked. The determination of the MIC value is the concentration at which no bacterial growth begins to occur in the first line. In the presence or absence of MtThe effect of bacteria on ampicillin MIC values is shown in Table 1. As is clear from Table 1, the MIC of ampicillin against E.coli with Cd treatment was 4. mu.g.mL^-1The MIC of ampicillin on E.coli under Mt-Cd treatment was 2. mu.g.mL^-1It can be concluded that the presence of Mt significantly reduces the ampicillin resistance of the bacteria.

And further verifying the RNA-Seq sequencing result by adopting a real-time fluorescent quantitative PCR technology. The differences in the expression of ampicillin resistance gene in E.coli in the presence or absence of Mt (montmorillonite) are shown in Table 2. From Table 2, it can be seen that the fold difference in the expression of the ampicillin resistance gene mrdA under Cd treatment was 1.67, while the fold difference under Mt-Cd treatment was 1.17; the fold differences in the expression of the ampicillin resistance genes mrcA, dacB and dacD under Cd treatment were 1.28, 1.03 and 1.11, whereas the fold differences under Mt-Cd treatment were not significant. Thus, it was found that the presence of Mt significantly suppressed the expression of the ampicillin resistance gene.

Example 5

Verifying the influence of Cd induction E.coli on the expression of tetracycline ARGs in the presence of Mt.

The strain after 12 hours of overnight activation was adjusted to 2.5MCF (1MCF ═ 3x 10) using a bacterial turbidimeter⁸ CFU·mL^-1) To ensure that the number of bacteria used in subsequent experiments is equal. The activated strains were added to Mt (12 gL) at a ratio of 1:100, respectively^-1) And 30mL Cd without Mt present²⁺The concentration was 16. mu.g/mL^-1Culturing for three generations in fresh LB culture medium (with sub-bacteriostasis concentration) for 24h each generation, wherein the culture conditions are 37 ℃, pH 7.0 and rotation speed of 200 rpm. Then transferred to Cd under the same conditions²⁺The concentration was 32. mu.g/mL^-1Subcultured 3 times in medium for 24h each time, and so on, and transferred to 64. mu.g.mL^-1、96μg·mL^-1、128μg·mL^-1Each passage was 3 times in the medium, each time for 24h, and the whole process was 15 days.

The test bacteria were diluted with MHB medium and dispersed to contain 0.5. mu.g.mL^-1、1μg·mL^-1、2μg·mL^-1、4μg·mL^-1、8μg·mL^-1、16μg·mL^-1、32μg·mL^-1、64μg·mL^-1、128μg·mL^-1A 96-well plate with tetracycline concentration, and the number of bacteria per well is ensured to be 10⁴And (4) CFU. Subsequently, the 96-well plate was placed in a constant temperature incubator (37 ℃) and incubated for 24 hours, and then the results were checked. The determination of the MIC value is the concentration at which no bacterial growth begins to occur in the first line. The effect of bacteria on the MIC values of tetracycline in the presence or absence of Mt is shown in Table 1. As can be seen from Table 1, the MIC of tetracycline to E.coli under Cd treatment was 8. mu.g.mL ^-1The MIC of tetracycline to Escherichia coli under the treatment of Mt-Cd was 4. mu.g.mL^-1It can be concluded that the presence of Mt significantly reduces the resistance of the bacteria to tetracycline.

And further verifying the RNA-Seq sequencing result by adopting a real-time fluorescent quantitative PCR technology. The differences in the expression of the tetracycline resistance gene by E.coli in the presence or absence of Mt are shown in Table 2. From Table 2, it can be seen that the fold difference in the expression of the tetracycline resistance gene accC under Cd treatment was 1.49, while the fold difference under Mt-Cd treatment was 1.33. Therefore, it is known that the expression of the tetracycline resistance gene is significantly suppressed by the presence of Mt.

Example 6

The effect of Cd-induced E.coli on the expression of erythromycin ARGs was verified in the presence of Mt.

The strain after 12 hours of activation was adjusted to 2.5MCF (1MCF ═ 3x 10) using a bacterial turbidimeter⁸CFU·mL^-1) To ensure that the number of bacteria used in subsequent experiments is equal. The activated strains were added to Mt (16 gL) at a ratio of 1:100, respectively^-1) And 30mL Cd without Mt present²⁺The concentration was 16. mu.g/mL^-1Culturing for three generations in fresh LB culture medium (with sub-bacteriostasis concentration) for 24h each generation, wherein the culture conditions are 37 ℃, pH 7.0 and rotation speed of 200 rpm. Then transferred to Cd under the same conditions²⁺The concentration was 32. mu.g/mL^-1Subcultured 3 times in medium for 24h each time, and so on, and transferred to 64. mu.g.mL ^-1、96μg·mL^-1、128μg·mL^-1The culture medium is subcultured for 3 times, each time for 24 hours, and the whole process is 15 days.

The test bacteria were diluted with MHB medium and dispersed to contain 0.5. mu.g.mL^-1、1μg·mL^-1、2μg·mL^-1、4μg·mL^-1、8μg·mL^-1、16μg·mL^-1、32μg·mL^-1、64μg·mL^-1、128μg·mL^-196-well plates with erythromycin concentration and a bacterial count of 10 per well⁴And (4) CFU. Subsequently, the 96-well plate was placed in a constant temperature incubator (37 ℃) and incubated for 24 hours, and then the results were checked. The determination of the MIC value is the concentration at which no bacterial growth begins to occur in the first line. The effect of bacteria on erythromycin MIC values in the presence or absence of Mt is shown in Table 1. As can be seen from Table 1, the MIC of erythromycin to E.coli in Cd treatment was 4. mu.g.mL^-1The MIC of tetracycline to Escherichia coli under the treatment of Mt-Cd was 2. mu.g.mL^-1It can be concluded that the presence of Mt significantly reduces the resistance of the bacteria to erythromycin.

And further verifying the RNA-Seq sequencing result by adopting a real-time fluorescent quantitative PCR technology. The differences in the expression of erythromycin resistance genes in E.coli in the presence and absence of Mt are shown in Table 2. From Table 2, it can be seen that the fold difference of the expression of the erythromycin resistance gene suhB under Cd treatment is 2.86, while the fold difference under Mt-Cd treatment is not obviously changed. Therefore, it is known that the presence of Mt significantly suppresses the expression of the erythromycin resistance gene.

Example 7

Effect of Cd-induced e.coli on chloramphenicol ARGs expression in the presence of Mt.

The strain after 12 hours of activation was adjusted to 2.5MCF (1 MCF-3 x 10) using a bacterial turbidimeter⁸CFU·mL^-1) To ensure that the number of bacteria used in subsequent experiments is equal. The activated strains were added to Mt (16 gL) at a ratio of 1:100, respectively^-1) And 30mL Cd without Mt present²⁺The concentration was 16. mu.g/mL^-1Culturing for three generations in fresh LB culture medium (with sub-bacteriostasis concentration) for 24h each generation, wherein the culture conditions are 37 ℃, pH 8.0 and rotation speed of 200 rpm. Then transferred to Cd under the same conditions²⁺The concentration was 32. mu.g/mL^-1Subcultured 3 times in medium for 24h each time, and so on, and transferred to 64. mu.g.mL^-1、96μg·mL^-1、128μg·mL^-1Each passage was 3 times in the medium, each time for 24h, and the whole process was 15 days.

The test bacteria were diluted with MHB medium and dispersed to contain 0.5. mu.g.mL^-1、1μg·mL^-1、2μg·mL^-1、4μg·mL^-1、8μg·mL^-1、16μg·mL^-1、32μg·mL^-1、64μg·mL^-1、128μg·mL^-196-well plates with chloramphenicol concentration, and the number of bacteria per well was guaranteed to be 10⁴And (4) CFU. Subsequently, the 96-well plate was placed in a constant temperature incubator (37 ℃) and incubated for 24 hours, and then the results were checked. The determination of the MIC value is the concentration at which no bacterial growth begins to occur in the first line. The effect of bacteria on chloramphenicol MIC values in the presence or absence of Mt is shown in Table 1. As can be seen from Table 1, the MIC of chloramphenicol for E.coli under Cd treatment was 4. mu.g.mL^-1The MIC of chloramphenicol to E.coli under Mt-Cd treatment was 2. mu.g.mL ^-1It can be concluded that the presence of Mt significantly reduces the resistance of the bacteria to chloramphenicol.

And (3) further verifying the RNA-Seq sequencing result by adopting a real-time fluorescent quantitative PCR technology. The differences in expression of chloramphenicol resistance gene in E.coli in the presence and absence of Mt are shown in Table 2. From Table 2, it can be seen that the fold difference in the expression of the chloramphenicol resistance gene tktA under Cd treatment was 1.64, whereas the fold difference under Mt-Cd treatment was not significantly changed. Therefore, it was found that the presence of Mt significantly suppressed the expression of the chloramphenicol resistance gene.

Meanwhile, the expression differences of the genes related to the multi-drug resistance/multi-antibiotic resistance/antibiotic response of the Escherichia coli (such as mdtQ, marB/R, arnA/B/C/D and the like) and a large number of toxin efflux related genes (such as emrA, mprA, ydhC, marA and the like) are shown in Table 2. As can be seen from Table 2, the expression fold difference of the genes of the Mt-Cd treatment group is lower than that of the Cd treatment group. Therefore, the expression of genes related to multidrug resistance/multiple antibiotic resistance/antibiotic response of Escherichia coli and a large number of toxin efflux related genes can be inhibited by the existence of Mt.

TABLE 1 comparison of MIC before and after Induction for different treatment groups

Cd treatment in Table 1 indicates Cd alone²⁺Performing induction treatment; Mt-Cd treatment is shown in Cd²⁺Montmorillonite is added for treatment under induction; representation of blank processing Escherichia coli only, without Cd²⁺Neither induction treatment added Mt.

TABLE 2 partial Differential Expression Genes (DEGs) associated with Cd-induced bacterial antibiotic resistance in the presence or absence of Mt

^aCd treatment (treatment 1) and Mt-Cd treatment (treatment 2) samples were varied from control sample e.coli by fold based on log2 gene abundance ratio.^bBy "-" is meant that there was no significant difference between the treated sample and the control.

The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.

Claims

1. the application of a reagent in suppressing bacteria in the expression of antibiotic resistance gene under metal induction, is characterized in that, described reagent comprises montmorillonite;

The concentration of described montmorillonite is 8-16gL ^-1 ;

The pH of the reagent is 6.0-8.0;

The metal induction is a Cd ²⁺ ion-induced environment;

The concentration of the Cd ²⁺ ions is 16-128 μg∙mL ^-1 ;

The preparation of the reagent includes the following steps:

(1) Add montmorillonite into water, then centrifuge, remove the supernatant, take the precipitate, then add the precipitate back to the water, repeat the steps of centrifugation until the supernatant is clear, dry, sieve, and sterilize at high temperature , to obtain pretreated montmorillonite;

(2) adding the pretreated montmorillonite in step (1) into a solvent and mixing evenly to obtain the reagent for inhibiting the expression of antibiotic resistance genes of bacteria induced by metals;

The rotating speed of the centrifugal treatment in step (1) is 8000-10000rpm; the drying temperature in step (1) is 55-65°C; the sieve size of the sieving in step (1) is 200 meshes;

The temperature of the high-temperature sterilization treatment is 100-120° C., and the time of the high-temperature sterilization treatment is 30-60 minutes;

The solvent in step (2) is deionized water;

The bacteria are wild-type Escherichia coli ATCC25922; the antibiotics are ampicillin, tetracycline, erythromycin or chloramphenicol.